Tan-1057 derivatives

ABSTRACT

The invention relates to novel natural product derivatives of the formula (I), to processes for their preparation, to pharmaceutical compositions comprising them and to their use in the treatment of disorders in humans or animals:                    
     in which R 1 , D, X, Y and Z are defined as in claim 1.

The present invention relates to novel natural product derivatives, toprocesses for their preparation, to pharmaceutical compositionscomprising them and to their use in the treatment of disorders in humansor animals.

EP-A-0339596 discloses antibiotics of the formula

which are obtained by cultivating a microorganism of the genusFlexibacter.

Specifically, this publication also describes the following compounds

in which the carbon atom A has the S configuration (TAN-1057A) or the Rconfiguration (TAN-1057B). Y. Funabashi et al.; Tetrahedron 49, 13, 1993describe the chemical and structural characterization of TAN-1057A andTAN-1057. B. N. Katayama et al.; J. Antibiotics, 46, 606, 1993 reportabout the taxonomy of TAN-1057-producing organisms and the biologicalproperties of TAN-1057. Total syntheses of TAN-1057 compounds werepublished by C. Yuan and R. M. Williams in J. Am. Chem. Soc.; 119,11777, 1997 and A. de Meijere et al. in Eur. J. Org. Chem. 1998, 777.First derivatives of TAN-1057 compounds were described by R. M. Williamsin J. Antibiotics; 51, 189, 1998. However, most of the derivatizationsrelate to the cyclic amidinourea moiety of the molecule. Thus, forexample, derivatives of the type

in which R represents Ac, COPh, COOMe, SO₂Me and CO₂CH₂Ph are described.

WO 99/07685, which was published after the date of priority of thepresent application, discloses derivatives, acylated at the cyclicamidinourea moiety of the molecule and additionally phosphorylated, ofthe general formula:

in which R₂ represents

Only two derivatizations (J. Antibiotics; 51, 189, 1998) relate to the(S)-β-homoarginine moiety:

However, these derivatizations resulted in a complete loss of biologicalactivity.

It was the object of the inventors of the present invention tosynthesize further derivatives of the TAN-1057 compounds to investigatetheir biological and/or pharmacological actions. After overcomingdifficult synthetic problems, the inventors succeeded in synthesizingfurther novel compounds which are derivatized in the (S)-β-homoargininemoiety of TAN 1057, using a novel, generally applicable process, whichcompounds, surprisingly, have considerably lower toxicity, withcomparable activity.

Accordingly, the present invention provides compounds of the generalformula:

in which

R¹ represents hydrogen or (C₁-C₆)alkyl,

X represents a group of the formula —(CH₂)_(m)—, in which m is 0, 1 or2,

D is selected from groups of the formulae D₁ to D₃

 in which R² represents hydrogen or hydroxyl,

R³ represents hydrogen, or

R² and R³ together form an oxo group,

Y represents a straight-chain or branched (C₁-C₅)alkanediyl group inwhich optionally one carbon atom may be replaced by —O— or —NH— andwhich may optionally be substituted by hydroxyl or oxo, or represents agroup of the formulae below

 in which r and s are identical or different and are 0, 1 or 2,

Z represents a group selected from groups of the formulae

 in which R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴,R¹⁵, R¹⁶, R¹⁷, R¹⁸ and R¹⁹ in each case independently of one another areselected from the group consisting of hydrogen, (C₁-C₆)alkyl,(C₁-C₄)alkanoyl, t-butoxycarbonyl, benzyloxycarbonyl and benzyl,

Q represents oxygen or sulphur and

pis 1, 2 or 3 and

Het represents a 5- or 6-membered heteroaromatic group having 1 to 4nitrogen atoms,

except for compounds in which

R¹ represents methyl, m is 1, D represents D₁, Y represents —(CH₂)₃— andZ represents a group of the formula

 and pharmaceutically acceptable salts thereof.

The case corresponding to TAN 1057A/B, in which R¹ represents methyl, mis 1, D represents D₁, Y represents —(CH₂)₃— and Z represents a group ofthe formula

which is excluded from the compounds claimed according to the invention,corresponds to the case in which D represents D₂, R² and R³ representhydrogen, R represents methyl, m is 1, Y represents —(CH₂)₂— and Zrepresents a group of the formula

which, as a consequence, is likewise excluded from the compoundsaccording to the invention.

The present invention preferably provides compounds of the generalformula:

in which R¹ represents hydrogen or (C₁-C₆)alkyl,

X represents a group of the formula —(CH₂)_(m)—, in which m is 0, 1 or2,

D is selected from groups of the formulae D₁ to D₃

 in which R² represents hydrogen or hydroxyl,

R³ represents hydrogen, or

R² and R³ together form an oxo group,

Y represents a straight-chain or branched (C₁-C₅)alkanediyl group inwhich optionally one carbon atom may be replaced by —O— or —NH— andwhich may optionally be substituted by hydroxyl or oxo, or represents agroup of the formulae below

 in which r and s are identical or different and are 0, 1 or 2,

Z represents a group selected from groups of the formulae

 in which R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶,R¹⁷, R¹⁸ and R¹⁹ in each case independently of one another are selectedfrom the group consisting of hydrogen, (C₁-C₆)alkyl, (C₁-C₄)alkanoyl,t-butoxycarbonyl, benzyloxycarbonyl and benzyl,

Q represents oxygen or sulphur and

p is 1, 2 or 3 and

Het represents a 5- or 6-membered heteroaromatic group having 1 to 4nitrogen atoms,

except for compounds in which

R¹ represents methyl, m is 1, D represents D₁, Y represents —(CH₂)₃— andZ represents a group of the formula

 (corresponds to the case in which D represents D₂, R² and R³ representhydrogen, R¹ represents methyl, m is 1, Y represents —(CH₂)₂— and Zrepresents a group of the formula

 and pharmaceutically acceptable salts thereof.

m in the group of the formula —(CH₂)_(m)— for X is preferably 1 or 2.Accordingly, the group of the formula —(CH₂)_(m)— for X preferablyincludes a methylene or an ethylene (ethane-1,2-diyl) group. X isparticularly preferably a methylene group.

A straight-chain or branched (C₁-C₅)alkanediyl group in which optionallyone carbon atom may be replaced by —O— or —NH— and which, additionally,may optionally be substituted by hydroxyl or oxo, in the definition of Yincludes, for example, straight-chain (C₁-C₅)alkanediyl groups, such asmethylene, ethylene, propane-1,3-diyl, butane-1,4-diyl andpentane-1,5-diyl. Preference is given to straight-chain(C₁-C₄)alkanediyl groups.

A straight-chain or branched (C₁-C₅)alkanediyl group in which one carbonatom is replaced by —O— or —NH— and which, additionally, may optionallybe substituted by hydroxyl or oxo, in the definition of Y includes, forexample, groups of the formulae:

Here, either side of the groups may be attached to Z.

The groups of the formulae

for Y include, for example, symmetrical radicals in which r and s areidentical, or asymmetrical radicals; symmetrical radicals in which r ands are 0, i.e. 1,2-phenylene, 1,3-phenylene and 1,4-phenylene, beingpreferred. Particular preference is given to 1,3- or m-phenylene.

In the general formula (I), Z is selected from groups of the formulae

in which R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷,R¹⁸ and R¹⁹ in each case independently of one another are selected fromthe group consisting of hydrogen, (C₁-C₆)alkyl, (C₁-C₄)alkanoyl,t-butoxycarbonyl, benzyloxycarbonyl and benzyl,

Q represents oxygen or sulphur and

p is 1, 2, or 3 and

Het represents a 5- or 6-membered heteroaromatic group having 1 to 4nitrogen atoms.

Among these groups for Z

R⁴, R⁵, R⁶, R⁷ and R¹⁷ are as defined above are preferred.

Z is particularly preferably a group of the formula

in which R⁴, R⁵, R⁶ and R⁷ are as defined above, preferably hydrogen.

In the group of the formula

for Z, Het is advantageously selected from the group consisting of thegroup of the formulae:

Here, the azoles, i.e. the five-membered unsaturated heterocyclic ringsystems having 1 to 4 nitrogen atoms, can be attached either via acarbon or via a nitrogen atom.

Among these radicals, six-membered heteroaromatics having 1 to 3nitrogen atoms are preferred.

Particularly preferably, Het represents pyridyl, including 2-pyridyl,3-pyridyl and 4-pyridyl, 2-pyridyl being particularly preferred.

Very particularly preferably, Z represents

In a further preferred embodiment, Z represents a group of the formula

in which R₁₈ and R¹⁹ are as defined above.

(C₁-C₆)Alkyl in the above definitions of R¹ and R⁴ to R¹⁹ represents astraight-chain or branched alkyl group having 1 to 6 carbon atoms suchas, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl,tert-butyl, sec-butyl, iso-butyl, pentyl and hexyl, preference beinggiven to (C₁-C₄)alkyl groups and particular preference being given tomethyl.

R⁴ to R¹⁹ preferably represent hydrogen.

(C₁-C₄)Alkanoyl in the definition of R⁴ to R¹⁹ represents, for example,formyl, acetyl, propionyl and butanoyl, preference being given to formyland acetyl.

Q preferably represents oxygen.

p is preferably 1 or 2.

In a preferred embodiment of the invention, the group D represents

In a further preferred embodiment of the invention, the group Drepresents

in which R² and R³ are as defined above and are preferably hydrogen.

In a preferred embodiment of the invention, the group D represents

Among these, preference is given to the groups D₁ and D₂ and particularpreference is given to D₁. Preference is furthermore given to the casein which both R¹ and R² in D₂ are hydrogen.

Suitable pharmaceutically acceptable salts of the compounds of thegeneral formula (I) can be conventional non-toxic salts including, forexample, salts with mineral acids, carboxylic acids or sulphonic acids.Such acid addition salts include organic acid salts (for exampleacetates, propionates, lactates, citrates, benzoates, trifluoroacetates,maleates, tartrates, fumarates, methanesulphonates, ethanesulphonates,benzenesulphonates, formates, toluenesulphonates,naphthalene-disulphonates, etc.) and inorganic acid salts (for examplehydrochlorides, hydrobromides, hydrolodides, sulphates, nitrates,phosphates, etc.).

As a consequence of double bonds and asymmetric carbon atoms, thecompounds of the general formula (I) can be present as stereoisomers,such as cis/trans isomers and configuration isomers, such as enantiomersor diastereomers; these isomers and mixtures thereof are included in thescope of the present invention.

The compounds according to the invention are prepared by reactingcompounds of the formula (II)

in which X, Y and Z are as defined above and D′ is selected from groupsof the formulae D′₁ to D′₃

in which R² and R³ are as defined above and A is a conventionallyprotected amino group, with compounds of the formula (III)

in which R¹ is as defined above, in the presence of coupling agents, ifdesired in the presence of bases, and the conventional protective groupin the protected amino group A is removed by methods known per se.

The compound of the formula (III) is synthesized using the method of V.V. Sokolov, S. I. Kozhushkov, S. Nikolskaya, V. N. Belov, M. Es-Sayed,A. de Meijere, Eur. J. Org. Chem. 1998, 777.

Compounds of the formula (II) used for the amide formation areappropriately protected amino acids which can be obtained, for example,by chain extension from the α-amino acids (cf. H. M. M. Bastiaans, A. E.Alewijnse, J. L. van der Baan, H. C. J. Ottenheijm, Tetrahedron Lett.1994, 35, 7659).

If the straight-chain or branched (C₁-C₅)alkanediyl group for Y containsfunctional groups, such as hydroxyl, keto, ester or amide functions,their introduction or synthesis is carried out by standard methods (see,for example, Houben-Weyl, Methoden der organischen Chemie [Methods oforganic chemistry], Volume XV/1 and 2.).

3-(Benzyloxycarbonylamino)-3-[3′-(N-benzyloxycarbonylguanidino)phenyl]-propionicacid, for example, is prepared according to Scheme 1 below.

Here, compound D (E. Proft, F.-J. Becker, J. prakt Chemie 1965, 30, 18),for example, is esterified with methanol in the presence of, forexample, concentrated sulphuric acid. In the two-phase systemdichloromethane/base, such as, for example, saturated aqueous sodiumbicarbonate solution, the free amino group is converted into the benzylcarbamate using benzyl chloroformate. The aromatic nitro group isreduced using tin(II) chloride. Subsequent reaction withbis(benzyloxycarbonyl) S-methylthiourea (W. Su, Synth. Commun. 1996, 26,407) in the presence of mercury(II) chloride and basic hydrolysis of themethyl ester gives the carboxylic acid I.

In the above reactions, it is also possible to use the generallyemployable solvents, acids and bases listed below in place of thosementioned here.

The compounds of the formula (II) in which D represents D₃ are prepared,for example, as illustrated in the following example:

Here:

Bn represents: —CH₂—Ph

Cbz represents: PhCH₂—O—C(O)—

Mes represents: CH₃S_(N) ₂—

Me represents: methyl

The reactions are preferably carried out under the conditions describedin the experimental section.

The radical Z in the acid component of the formula (II) is, inprinciple, synthesized by reacting an S-methyl thiourea derivative witha terminal amino group. This reaction is mediated by basic reagents,such as tertiary amines or sodium hydroxide, if appropriate in thepresence of mercury(II) chloride.

If Z is a carbamate group, the carbamate group is obtained by reactingthe terminal amino group with a chloroformic ester in the presence of abase (for example a tertiary amine or sodium hydroxide) (Scheme 3).Here, the acid function can be present in free form or blocked by anappropriate protective group (for example an alkyl ester).

Here, the substituents or substituent groups are as defined above, andHal represents halogen, such as fluorine, chlorine, bromine and iodine.

In the case that R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵,R¹⁶, R¹⁷, R¹⁸ and R¹⁹ in the above process are not hydrogen but(C₁-C₆)alkyl, (C₁-C₄)alkanoyl, t-butoxycarbonyl, benzyloxycarbonyl andbenzyl, the amino groups are alkylated by customary methods, for exampleusing alkylating agents, such as alkyl halides, sulphonic esters orsubstituted or unsubstituted dialkyl- or diarylsulphonates, such asmethyl iodide or dimethyl sulphate, and (C₁-C₄)alkanoyl,t-butoxycarbonyl, benzyloxycarbonyl and benzyl, which are conventionalamino protective groups, are introduced by customary methods (cf. T. W.Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis, 2ndedition, John Wiley and Sons, New York, 1991).

The conventional amino protective group in the conventionally protectedamino group A is advantageously removed using the methods describedbelow.

Suitable for use as coupling agents in the reaction of the compounds ofthe formula (II) and (III) are known reagents, such as, for example,O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) or bromo-tris-pyrrolidino-phosphoniumhexa-fluorophosphate (PyBroP), since their use ensures a smooth couplingwith high yields.

Suitable for use as conventional amino protective groups in theconventionally protected amino group A (i.e. the conventionallyprotected —NH₂ group) are customary amino protective groups used inpeptide chemistry. These preferably include: benzyloxycarbonyl,2,4-dimethoxybenzyloxycarbonyl, 4-methoxy-benzyloxycarbonyl,methoxycarbonyl, ethoxycarbonyl, tert.-butoxycarbonyl,allyl-oxycarbonyl, phthaloyl, 2,2,2-trichloroethoxycarbonyl,fluorenyl-9-methoxycarbonyl, formyl, acetyl, 2-chloroacetyl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, phthalimido,isovaleroyl or benzyloxymethylene, 4-nitrobenzyl, 2,4-dinitrobenzyl,4-nitrophenyl, 4-methoxyphenyl or triphenylmethyl, particular preferencebeing given to benzyloxycarbonyl.

The removal of the amino protective group in the conventionallyprotected amino group A is carried out by conventional methods (cf. T.W. Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis, 2ndedition, John Wiley and Sons, New York, 1991), namely, preferably,tert-butyloxycarbonyl using hydrochloric acid in dioxane,fluorenyl-9-methoxycarbonyl using piperidine and benzyloxycarbonyl byhydrogenolysis in the presence of catalysts such as, for example, withRancy nickel, palladium, palladium on carbon or platinum or preferablypalladium(II) chloride. Any amino protective groups present in group Z,such as, for example, benzyloxycarbonyl, can also be removed in thisreaction.

In the present invention, the reactions are carried out in inert organicsolvents which do not change under the reaction conditions. Theseinclude ethers, such as diethyl ether, 1,4-dioxane or tetrahydrofuran,halogenated hydrocarbons, such as dichloromethane, trichloromethane,carbon tetrachloride, 1,2-dichloroethane, trichloroethane ortetrachloroethane, hydrocarbons, such as benzene, toluene, xylene,hexane, cyclohexane or mineral oil fractions, alcohols, such asmethanol, ethanol or iso-propanol, nitromethane, dimethylformamide oracetonitrile. It is also possible to use mixtures of the solvents.Particular preference is given to dichloromethane, tetrahydrofuran,dioxane, methanol or dimethylformamide.

The reactions are generally carried out in a temperature range of from0° C. to 150° C., preferably from 0° C. to 70° C. The reactions can becarried out under atmospheric, elevated or under reduced pressure (forexample from 0.5 to 5 bar). In general, they are carried out underatmospheric pressure.

Suitable bases for the processes according to the invention are, ingeneral, sodium bistrimethylsilylamide or lithiumbistrimethylsilylamide, alkali metal hydroxides, such as sodiumhydroxide, lithium hydroxide or potassium hydroxide, sodium bicarbonate,sodium hydride or organic (trialkyl(C₁-C₆)amines) such as triethylamine,or heterocycles, such as 1,4-diazabicyclo[5.4.0]undec-7-ene (DBU),pyridine, diaminopyridine, methylpiperidine or N-methylmorpholine.Preference is given to lithium hydroxide, sodium bicarbonate, pyridine,diisopropylethylamine and triethylamine.

A suitable acid can include an organic acid (for example formic acid,acetic acid, propionic acid, trichloroacetic acid, trifluoroacetic acid,etc.), an inorganic acid (for example hydrochloric acid, hydrobromicacid, sulphuric acid, hydrogen chloride, hydrogen bromide, hydrogenfluoride, etc.) etc.

The natural product derivatives of the present invention haveinteresting pharmacological actions. In particular, the compounds of thepresent invention have antibacterial action, and they are thereforeeffective in the control of bacterial infections in humans and animals.Furthermore, the known compounds TAN-1057 A and B have the problem thatthe treated germs rapidly become resistant to these compounds, and it isthought that the compounds of the present invention may less rapidlylead to the development of resistance, whilst having comparableantibacterial activity.

TAN1057 A,B shows pronounced toxicity in the liver and cells of theimmune system, which virtually excludes a use of this substance for thetherapy of bacterial infections. Compared to TAN 1057 A,B, all compoundsof the invention have reduced toxicity, which may permit therapeuticaluse.

Determination of the Minimum Inhibitory Concentration (MIC)

The MIC was determined using the liquid dilution test. Overnightcultures of the test germs (S. aureus 133) in Isosensitest bouillon werediluted 1:1000 with foetal calf serum (FCS) or Isosensitest bouillon andincubated with dilutions of the test substances (successive dilutions of1:2).

Selection of TAN 1057 A,B-resistant Bacteria

Bacteria of the strain S. aureus 133 were incubated with differentconcentrations of TAN 1057 A,B for 24 hours. Bacteria which showedvisible growth at elevated TAN 1057 A,B concentrations were transferredinto new culture bottles with different TAN 1057 A,B concentrations andincubated again. This process was repeated for several days, selectingstep by step bacteria having increased resistance to TAN 1057 A,B. TheMIC of highly resistant S. aureus 133 bacteria was >100 μg/ml (initialMIC: <0.1 μg/ml). The bacteria from this selection series, having an MICof <0.1, 0.8, 25, 100 and >100 μg/ml, were used for tests. It is thuspossible to identify TAN 1057 A,B derivatives having antibacterialactivity against TAN 1057 A,B-resistant cells.

Toxicological Studies Description of the Methods

Compatibility tests of the exemplary compounds were carried out usingcultures of eukaryotic cells. Hepatocytes (HepG2) and murine macrophagecells (J774.A1) were used as indicator for organ-specific toxicity. Themurine macrophage cell line used is a particularly sensitive indicatorfor toxic effects. All derivatives tested showed lower toxicity than TAN1057 A,B.

Test with HepG2 Cells:

The vitality and functionality of human HepG2 liver cells were examinedafter treatment with exemplary compounds. In each case 2×10⁴−1×10⁵ cellswere incubated in RPMI 1640 (Whittacker) with 10% heat-inactivatedfoetal calf serum (Gibco) at 37° C. for 40-48 hours. The incubationvolume of 200 μl contained the test substances in concentrations of 10,2 and 0.4 μg/ml.

The vitality was examined after addition of 20 μl Alamar Blue(Biosource, Art. No. DAL 1100) by measuring the fluorescence(excitation: 544 nm, emission: 590 nm).

The functionality of the HepG2 cells was examined by measuring thesecretion of apo B100 and α2-macroglobulin after treatment. To this end,the protein content of the culture supernatants was determined by ELISAafter incubation for 40-48 hours. Apo B100 was bound using rabbit ahLDL(1 mg/ml) and quantified using peroxidase-conjugated monoclonalantibodies (ahLDL_POD), with addition of the substrate TMB/H₂O₂. Theoptical density was measured at 450 nm.

To determine α2-macroglobulin, the ELISA system from Biodesign (Art.No.H 45205M) was used. Quantification was likewise carried out using thesubstrate TMB/H₂O₂, and measuring the optical density at 450 nm.

Test With J774.A1 Cells:

In each case 1.2×104 cells were incubated at 37° C. for 24 hours. Testsubstances were added at different concentrations, and the cells wereincubated for another 72 hours. The cells were then treated with 50 μlof Neutral Red solution (Sigma, No. N 2889) for 2 hours, washed with PBSmedium and denaturated using an ethanol/glacial acetic acid mixture.

The cultures were measured in an Elisa reader at 540 nm and 630 nm. TheIC₅₀ values were extrapolated and indicate at which concentration thevitality of the cells, measured by the uptake of Neutral Red, is reducedto 50%, compared to untreated control cells.

Results

The cultures were incubated at 37° C. for 18-24 hours. In each case thelowest concentration of substance at which no visible bacterial growthtook place, was defined as MIC.

MIC in FCS (S.aureus; μg/ml) Example  1 0.2  2 50  3 100  4 0.4  5 0.1 6 0.8  7 0.8  8 1.6  9 1.6 10 3.2 11 3.2 12 6.3 13 12.5 14 12.5 15 10016 100 17 6.3 18 12.5 Comparative Example TAN 1057 A, B (1:1 mixture of0.1 the two epimers)

The S. aureus 133 isolates with medium and high resistance to TAN 1057A,B were tested in comparison to the derivatives of the invention. Inthe MIC test with the isolated which showed medium resistance to TAN1057 A,B (MIC to TAN 1057 A,B=0.8 μg/ml), Ex. 4 showed betterantibacterial activity (MIC to Example 4=0.2 μg/ml).

Test of Antibacterial Activity in vivo

Mice were infected with 1×10⁶ bacteria of the strain S. aureus 133 in 5%mucine (i.p.) and treated by intravenous administration of the testsubstances 30 minutes after the infection. Without treatment, allinfected animals died. The therapeutic doses for TAN 1057 A,B and thecompound of Example 4 at which all infected animals survived (=ED₁₀₀)was 1 mg/kg for both substances.

Up to the highest test concentration, the compounds of Examples 1 and 2show no inhibition in the vitality and functionality tests (IC₅₀>10μg/ml). In the α2-macroglobulin assay, an inhibition (IC₅₀ 7 μg/ml) wasfound for TAN 1057 A,B.

In the Neutral Red vitality test using J774.A1 cells, an IC₅₀ value of0.25 μg/ml was determined for TAN 1057 A,B. All derivatives of TAN 1057of the present invention tested show higher IC₅₀ values, indicating thein some cases considerably better compatibility of the compounds.

IC₅₀ values are listed in the table below:

Example IC₅₀ value TAN 1057 A,B (1:1 mixture of the two epimers) 0.25 13 2 6 3 40 4 25 5 6 6 5.5 7 50 8 1.8

Acute Toxicity of TAN 1057 A,B and the Compound of Example 4

The acute toxicity of the substances was determined by determining thedose at which 50% of the treated mice survive (=LD₅₀). Followingintraperitoneal administration of the test substances, the LD50 for TAN1057 A,B was 100 mg/kg. The LD50 for the compound of Example 4 was >400mg/kg.

This result reflects the considerably lower toxicity of the compoundsaccording to the invention. However, as described above, the therapeuticactivity in vivo of TAN 1057 A,B and the compound of Example 4 wascomparable.

The compounds of the general formulae (I) according to the inventionhave a broad antibacterial spectrum, specifically against gram-positivegerms and some gram-negative bacteria, and also against corynebacteria.Owing to these properties, they can be used as chemotherapeuticallyactive compounds in human and veterinary medicine.

With their aid, it is possible to control gram-positive germs (withparticularly good action against staphylococci, includingmethillicin-resistant Staph. Aureus), gram-negative bacteria (forexample Moraxella catarrahlis) and also corynebacteria and to prevent,improve and/or heal disorders caused by these pathogens.

They are highly suitable for the prophylaxis and chemotheraphy of localand systemic infections caused by such pathogens, in human andveterinary medicine.

The present invention includes pharmaceutical preparations which, inaddition to non-toxic inert pharmaceutically suitable carriers orexcipients, comprise one or more compounds according to the invention,or which consist of one or more active compounds according to theinvention, and also processes for preparing these preparations.

The active compound(s) can, if appropriate, also be present inmicroencapsulated form, in one or more of the abovementioned carriers.

In the abovementioned pharmaceutical preparations, the therapeuticallyactive compounds should preferably be present in a concentration ofabout 0.1 to 99.5, preferably about 0.5 to 95, % by weight of the totalmixture.

The abovementioned pharmaceutical preparations may, in addition to thecompounds according to the invention, also comprise otherpharmaceutically active compounds.

In general, it has been found to be advantageous, both in human and inveterinary medicine, to administer the active compound(s) according tothe invention in total amounts of from about 0.5 to about 500,preferably 5 to 100, mg/kg of body weight per 24 hours, if appropriatein the form of a plurality of individual administrations, to obtain thedesired results. An individual administration preferably contains theactive compound(s) according to the invention in amounts of from about 1to about 80, in particular 3 to 30, mg/kg of body weight.

To widen the activity spectrum and to achieve an increase in activity,it is also possible to combine the compounds according to the inventionwith other antibiotics.

EXAMPLES

Abbreviations:

DMF N,N-dimethylformamide

HATU O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate

PE petroleum ether (special benzine, b.p. 40-80° C.)

THF tetrahydrofuran

Example 1

(3′S,5R,S)-5-[N-Methyl-N-(3′-amino-7′-guanidinoheptanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

A solution of 40 mg (0.216 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one, 131 mg(0.261 mmol) of(3S)-3-benzyloxycarbonylamino-7-[bis-(N-benzyloxycarbonyl)guanidino]heptanoicacid, 80 mg (0.432 mmol) ofO-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) and 53 mg (0.432 mmol) ofdiisopropylethylamine in 5 ml of DMF is stirred at 23° C. for 16 h. Thesolvent is then removed under reduced pressure. The residue is stirredwith 2 M hydrochloric acid. The aqueous phase is decanted off from theresidue. The residue is taken up in dichloromethane. The organic phaseis extracted twice with 2 M hydrochloric acid, dried with sodiumsulphate and concentrated under reduced pressure. This gives 155 mg(93%) of the coupling product as a white solid [MS (ESI): 772 (M+H)⁺].The solid is dissolved in 30 ml of methanol. The solution is admixedwith 65 mg (0.369 mmol) of palladium(II) chloride and stirred under anatmosphere of hydrogen (atmospheric pressure) for 4 h. The solution isfiltered off and concentrated under reduced pressure. The resultingresidue is stirred with diethyl ether and filtered off. This gives thetitle compound as a beige solid (80 mg, 93%). ¹H-NMR (400 MHz, CD₃OD):1.54 (m, 2H), 1.66 (m, 2H), 1.75 (m, 2H), 2.76 (dd, 1H), 2.98 (td, 1H),3.17+3.36 (s, 3H), 3.21 (dd, 2H), 3.59 (m, 1H), 3.89 (m, 1H), 4.03 (m,1H), 5.16 (m, 1H). MS (ESI) 370 (M+H)⁺.

Example 2

(3′RS,5RS)-5-{N-Methyl-N-[3′-amino-3′-(3-guanidylphenyl)propionyl]amino}-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

(Compounds D to I Refer to Scheme 1)

A solution of 79.5 g (0.378 mol) D, 3-amino-3-(3-nitrophenyl)propionicacid, in 21 of methanol is admixed with 200 ml of sulphuric acid(conc.). The solution is heated at boiling point for 1 h. Most of themethanol is removed under reduced pressure and the residue is pouredinto ice-water. Using sodium carbonate, the pH is adjusted to 8. Theaqueous phase is extracted with dichloromethane. Drying of the organicphase and removal of the solvent under reduced pressure gives the esterE, methyl 3-amino-3-(3-nitrophenyl)propionate, (52.8 g, 62%) as a whitesolid. 10 g (44.6 mmol) of E are dissolved in 20 ml of DMF. The solutionis admixed with 12.3 g (88.2 mmol) of potassium carbonate, and 15.2 g(88.2 mmol) of benzyl chloroformate are added dropwise. The mixture isstirred at 23° C. for 2 h. The mixture is diluted with 200 ml of tolueneand washed three times with water, and the organic phase is dried usingsodium sulphate and concentrated under reduced pressure to give thecolourless oil of methyl3-benzyloxycarbonylamino-3-(3-nitrophenyl)propionate (12.6 g, 79%). MS(DCI/NH₃): 376 (M+NH₄)⁺.

At 23° C., a solution of 4 g (11.1 mmol) of compound F, methyl3-benzyloxycarbonylamino-3-(3-nitrophenyl)propionate in 20 ml of ethanolis added dropwise to a solution of 12.6 g (55.8 mmol) of tin(II)chloride dihydrate. The mixture is stirred at a bath temperature of 80°C. for 30 minutes, and most of the ethanol is then removed under reducedpressure. The residue is partitioned between water and ethyl acetae. ThepH of the aqueous phase is adjusted to 8 by adding sodium bicarbonate.The mixture is filtered through a 5-cm-layer of Celite, which is washedwith ethyl acetate. The phases are separated and the aqueous phase isthen extracted three times with ethyl acetate. The organic phase isdried (sodium sulphate). Removal of the solvent under reduced pressuregives 3.8 g of a colourless oil G, methyl3-benzyloxycarbonylamino-3-(3-aminophenyl)propionate, which stillcontains tin salts. ¹H-NMR (200 MHz, CDCl₃): 2.87 (dd, 2H), 3.61 (s,3H), 5.08 (m, 1H), 5.11 (s, 2H), 6.60 (m, 3 H), 7.11 (t, 1H), 7.35 (m, 5H). MS (DCI/NH₃): 346 (M+NH₄)⁺.

A solution of 0.92 g (2.8 mmol) of G, methyl3-benzyloxycarbonylamino-3-(3-aminophenyl)-propionate and 1.0 g (2.8mmol) of bis(benzyloxycarbonyl) S-methyl thiourea in 10 ml of DMF isadmixed with 1.56 ml (11.2 mmol) of triethylamine and 0.83 g (3.1 mmol)of mercury(II)chloride. The mixture is stirred at 23° C. for 1 h,diluted with 100 ml of ethyl acetate and filtered through a 5-cm-layerof Celite. The organic phase is washed with saturated aqueous sodiumbicarbonate solution and dried (sodium sulphate) and the volatilecomponents are removed under reduced pressure. The residue ischromatographed over silica gel (dichloromethane:ethyl acetate=1:1).This gives 1.5 g (84%) of H, methyl3-benzyloxycarbonyl-amino-3-[3-(N,N′-bisbenzyloxycarbonylguanidino)phenyl]-propionateas a white solid. ¹H-NMR (200 MHz, CDCl₃): 2.86 (m, 2H), 3.60 (s, 3H),5.17 (m, 7H), 7.09 (d, 1H), 7.35 (m, 17H), 7.65 (d, 2H), 10.26 (s, br,1H), 11.90 (s, br, 1H). MS (ESI): 639 (M+H)⁺.

A solution of 500 mg (0.78 mmol) of compound H, methylbenzyloxycarbonylamino-3-[3-(N,N′-bisbenzyloxycarbonylguanidino)phenyl]propionate,in 7 ml of THF and 3.5 ml of water is admixed with 66 mg (1.56 mmol) oflithium hydroxide monohydrate. The mixture is heated at boiling pointfor 16 h, and the solvent is then removed under reduced pressure. Theresidue is partitioned between water and ethyl acetate. The phases areseparated and the aqueous phase is then adjusted to pH 1 usingconcentrated hydrochloric acid. The aqueous phase is extracted twicewith ethyl acetate, and the organic phase is dried over sodium sulphateand concentrated. The residue is chromatographed over silica gel(dichloromethane/ethyl acetate gradient). This gives3-(benzyloxycarbonylamino)-3-[3-(N-benyloxycarbonyl-guanidino)phenyl]-propionicacid (I) (270 mg, 70%) as a colourless oil. ¹H-NMR (400 MHz, CD₃OD):2.78 (m, 2H), 5.05 (s, 2H), 5.11 (m, 1H), 5.31 (s, 2H), 7.37 (m, 14H).MS (ESI): 491 (M+H)⁺.

At 23° C., a solution of 20 mg (0.108 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one, 53 mg(0.108 mmol) of the acid 1,40 mg (0.216 mmol) ofO-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate (HATU) and 26 mg (0.216 mmol) ofdiusopropylethylamine in 5 ml of DMF is stirred for 16 h. The solvent isthen removed under reduced pressure. The residue is stirred with 2 Mhydrochloric acid. The aqueous phase is decanted off from the residue.The residue is taken up in dichloromethane. The organic phase isextracted twice with 2 M hydrochloric acid, dried using sodium sulphateand concentrated under reduced pressure. This gives 70 mg (98%) of thecoupling product as a white solid [MS (ESI): 658 (M+H)⁺]. This isdissolved in 10 ml of methanol. The solution is admixed with 38 mg(0.213 mmol) of palladium(II) chloride and stirred under an atmosphereof hydrogen (atmospheric pressure) for 4 h. The solution is filtered andconcentrated under reduced pressure. The residue is triturated withdiethyl ether. The ether is decanted off and the residue is dried underreduced pressure, giving the title compound as a colourless oil (28 mg,57%). ¹H-NMR (400 MHz, CD₃OD): 2.81-3.34 (m, 7H), 3.86+3.99 (m, 1H),4.77+5.16 (m, 1H), 7.35-7.62 (m, 4H). MS (ESI) 390 (M+H)⁺.

Example 3

5-{N-Methyl-N-2′-[1-amino-2-(2-guanidinoethyl)cyclopropyl]acetamino}-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

The reactions are carried out in accordance with scheme 2 above, and thecompounds are referred to as J to S, in accordance with scheme 2.

At 0° C., a solution of 62.9 g (185 mmol) of J,2-(2-benzyloxyethyl)-1-chloro-1-(1,2,2-trichlorovinyl)cyclopropane, in50 ml of anhydrous methanol is slowly added dropwise with stirring to afreshly prepared solution of 34.0 g of sodium (1.48 mol, 8 equivalents)in 250 ml of methanol. The mixture is then heated at reflux for 2 d.After cooling to room temperature, the mixture is admixed with 700 ml ofwater and extracted with diethyl ether. The combined organic phases areconcentrated under reduced pressure and the residue is taken up in 300ml of methanol. 25 ml of 10 per cent hydrochloric acid are added and thereaction mixture is stirred for 45 min. The mixture is then admixed with300 ml of saturated sodium bicarbonate solution and extracted withdiethyl ether. The ether extracts are dried over calcium chloride.Removal of the solvent under reduced pressure gives a dark-brown oil.Filtration through 300 g of silica gel (PE:diethyl ether 10:1) gives24.4 g (47%) of K methyl[2-(2-benzyloxyethyl)cyclo-propylidene]chloroacetate in the form of apale yellow liquid as a mixture of two diastereomers in the ratio of1:1.6.-¹³C-NMR (62.9 MHz, CDCl₃, additionally DEPT), isomer A: δ=11.6(−), 20.3 (+), 31.6 (−), 52.8 (+), 68.8 (−), 72.9 (−), 115.3(C_(quart)), 127.49 (+), 127.53 (+), 128.3 (+), 138.3 (C_(quart)), 143.8(C_(quart)), 162.4 (C_(quart)). -isomer B: δ=15.6 (−), 16.1 (+), 31.5(−), 52.8 (+), 69.3 (−), 73.0 (−), 114.6 (C_(quart)), 127.49 (+), 127.53(+), 128.3 (+), 143.3 (C_(quart)), 162-7 (C_(quart)). MS (70 eV), m/z(%): 280 (<1) [M⁺], 245 (1) [M⁺—Cl], 189 (2) [M⁺—CH₂Ph], 91 (100)[CH₂Ph⁺]. —C₁₅H₁₇ClO₃ (280.8): calc. C, 64.17 H, 6.10; found C, 63.24,H, 5.97.

A solution of 11.98 g (42.7 mmol) of K, methyl[2-(2-benzyloxyethyl)cyclo-propylidenelchloroacetate, in 100 ml ofanhydrous methanol is slowly mixed with 8.42 g (42.7 mmol) of anhydrousN,N-dibenzylamine. The solution is stirred at room temperature for 16 hand then concentrated under reduced pressure. Column chromatography over300 g of silica gel (PE:diethyl ether 9:1) gives 15.71 g (77%) of L,methyl(E)-2-[2-(benzyloxyethyl)-1-(N,N-dibenzyl-amino)cyclopropyl]-2-chloroacetate,(pale-yellow oil) as a mixture of two diastereomers in the ratio1:1.6.-¹³C-NMR (62.9 MHz, CDCl₃, additionally DEPT), isomer A: δ=23.6(−), 29.2 (+), 29.4 (−), 51.1 (C_(quart)), 52.8 (+), 56.6 (−), 62.1 (+),69.8 (−), 72.8 (−), 126.5 (+), 126.6 (+), 127.6 (+), 128.3 (+), 128.7(+), 128.8 (+), 138.4 (C_(quart)), 139.7 (C_(quart)), 169.1 (C_(quart)).-isomer B: δ=23.2 (−), 26.0 (+), 29.5 (−), 50.4 (C_(quart)), 52.9 (+),56.6 (−), 64.3 (+), 69.8 (−), 72.9 (−), 126.5 (+), 126.6 (+), 127.6 (+),128.3 (+), 128.7 (+), 128.8 (+), 138.4 (C_(quart)), 139.0 (C_(quart)),169.5 (C_(quart)). MS (70 eV), m/z (%): 442 (<1) [M⁺—Cl], 386 (<1)[M⁺—CH₂Ph], 91 (100) [CH₂Ph⁺]. —C₂₉H₃₂ClNO₃ (478.0): calc. C, 72.87, H,6.75; found C, 72.53, H, 6.84.

An autoclave is charged with 100 ml of methanol and about 2.00 g ofpalladium on activated carbon (10% strength, 50% water), flushedrepeatedly with hydrogen and stirred at 4.5 bar for 30 min. A solutionof 8.86 g (18.5 mmol) of L, methyl (E)-2-[2-(benzyloxyethyl)-1-(N,N-dibenzylamino)cyclopropyl]-2-chloroacetate, in100 ml of methanol is added to the activated catalyst, and the mixtureis stirred at 4.5 bar and room temperature for 7 days. The catalyst isthen separated off by filtration through Celite and the filtrate isconcentrated under reduced pressure. The residue is suspended in 110 mlof saturated sodium carbonate solution and, with vigorous stirring andin an ice-bath, admixed with 4.51 g (1.43 equivalents) of benzylchloroformate and stirred at the same temperature for 5 h. Followingextraction with ethyl acetate and drying over magnesium sulphate, theproduct is purified by column chromatography over silica Igel (diethylether). This gives 3.73 g (66%) of N, methyl(E)-2-[1-amino-2-(hydroxyethyl)cyclo-propyl]acetate hydrochloride.-¹H-NMR (250 MHz, CDCl₃): δ=0.39 (m_(c), 1H), 1.00-1.30 (m, 3H),1.9-02.00 (m, 1H), 2.18 (d, J²=17.3 Hz, 1H,), 3.02 (d, J²=17.3 Hz, 1H),3.66 (s, 3H), 3.74 (m_(c), 2H), 5.05 (m_(c), 2H), 5.92 (s, 1H), 7.31(m_(c), 5H). -¹³C-NMR (62.9 MHz, CDCl₃, additionally DEPT): δ=18.0 (−),24.4 (+), 32.1 (−), 33.6 (C_(quart)), 37.4 (−), 51.5 (+), 61.9 (−), 66.9(−), 128.0 (+), 128.1 (+), 128.4 (+), 136.0 (C_(quart)), 157.1(C_(quart)), 172.6 (C_(quart)). -MS (70 eV), m/z (%): 307 (<1) [M⁺], 276(<1) [M⁺—OCH₃], 172 (8) [M⁺—OCOCH₂Ph], 91 (100) [CH₂Ph⁺].

A solution of 1.600 g (5.209 mmol) of N, methyl(E)-2-[1-amino-2-(hydroxyethyl)cyclopropyl]-acetate hydrochloride, in 40ml of dry dichloromethane is cooled to 0° C., admixed with 1.02 g (2.0equivalents) of triethylamine and 1.19 g (2.0 equivalents) ofmethanesulphonate chloride and stirred at the same temperature for 2 h.The solvent is removed under reduced pressure and the residue is takenup in 50 ml of ethyl acetate and washed with 40 ml of NaHCO₃ soln. Theaqueous phase is extracted with ethyl acetate. The organic phases aredried over magnesium sulphate and the solvent is removed under reducedpressure, giving 2.010 g (quantitative) of O, methyl(E)-2-[1-benzyloxycarbonylamino-2-(hydroxyethyl)cyclopropyl]-acetate, inthe form of a slightly yellowish solid. -¹H-NMR (250 MHz, CDCl₃): δ=0.48(m_(c), 1H), 1.05-1.20 (m, 2H), 1.65-1.90 (m, 2H), 2.52 (d, 1H), 2.60(d, 1H), 2.99 (s, 3H), 3.65 (s, 3H), 4.35 (m_(c), 2H), 5.02 (s, 2H),5.68 (s, 1H), 7.30 (m_(c), 5H). —C₁₇H₂₃NO₇S (385.4): calc. C, 52.98, H,6.01; found C, 53.08, H, 6.00.

2.010 g (5.209 mmol) of O, methyl(E)-2-[1-benzyloxycarbonylamino-2-hydroxyethyl)cyclopropyl]-acetate, aredissolved in 10 ml of dry DMF, admixed with 1.69 g (5.0 equivalents) ofsodium azide and stirred at room temperature for 4 d. The solvent isremoved under reduced pressure and the residue is then admixed with 100ml of water and extracted twice with ethyl acetate, the extract is driedover sodium sulphate and the solvent is removed under reduced pressure.Column chromatography over silica gel (PE:diethyl ether 1:1→DE) gives1.540 g (89%) of P, methyl(E)-2-[2-(azidoethyl)-1-(benzyloxycarbonylamino)cyclopropyl]acetate inthe form of a colourless oil. -¹H-NMR (250 MHz, CDCl₃): δ=0.48 (m_(c),1H), 1.05-1.26 (m, 2H), 1.49 (m_(c), 1H,), 1.75 (m_(c), 1H), 2.54 (d,J²=17 Hz, 1H), 2.69 (d, J²=17 Hz, 1H), 3.42 (m_(c), 2H), 3.68 (s, 3H),5.05 (s, 2H), 5.60 (br. s, 1H), 7.33 (m_(c), 5H). -¹³C-NMR (62.9 MHz,CDCl₃, additionally DEPT): δ=19.3 (−), 23.2 (+), 29.2 (−), 33.5 (C),36.6 (−), 50.8 (−), 51.8 (+), 66.5 (−), 128.0 (+), 128.5 (+), 128.5 (+),136.2 (C_(quart)), 155.8 (C_(quart)), 172.3 (C_(quart)). -MS (DCI, NH₃),m/z (%): 350 (100) [M⁺+NH₄], 333 (10) [M⁺+H]. —C₁₆H₂₀N₄O₄ (332.4): calc.C, 57.82, H, 6.07; found C, 57.54, H, 5.87.

At room temperature, a solution of 1.51 g (4.54 mmol) of P, methyl(E)-2-[2-(azidoethyl)-1-(benzyloxycarbonylamino)-cyclopropyl]acetate, in5 ml of THF is admixed with 1.19 g (1.0 equivalents) oftriphenylphosphine and 82 μl (4.54 mmol) of water and stirred for 24 h.The solvent is removed under reduced pressure. 10 ml of a mixture ofPE:diethyl ether=1:1 are added to the residue, and the mixture istreated in an ultrasonic bath until triphenylphosphine oxideprecipitates out. The latter is filtered off and washed repeatedly witha total of 100 ml of the solvent mixture. The filtrate is concentratedunder reduced pressure and the residue is then dissolved in 15 ml of DMFand admixed with 1.63 g (1.0 equivalents) of N,N′-bis(benzyloxycarbonyl)S-methyl isothiourea, 1.23 g (1.0 equivalents) of mercury(II) chlorideand 0.92 g (2.0 equivalents) of triethylamine. After 2 h of stirring,the mixture is filtered through Celite, which is then washed with 150 mlof diethyl ether. The solvents are removed under reduced pressure andthe residue is then taken up in 200 ml of dichloromethane and washedwith 100 ml of water. Following drying over magnesium sulphate, theproduct is purified by column chromatography over silica gel (diethylether). This gives 1.82 g (65%) of R, methyl(E)-2-{2-[N,N′-(bisbenzyloxycarbonyl)guanidino]ethyl-1-(benzyloxycarbonyl-amino)cyclopropyl]acetatein the form of a glass-like oil. -¹H-NMR (250 MHz, CDCl₃): δ=0.48(m_(c), 1H), 1.11 (m_(c), 2H), 1.63 (m_(c), 2H), 2.52 (d, J²=17.3 Hz,1H), 2.74 (d, J²=17.3 Hz, 1H), 3.40-3.80 (m, 2H), 3.66 (s, 3H),5.05-5.20 (m, 6H), 5.69 (s, 1H), 7.2-7.4 (m, 15H), 8.61 (br. s, 1H),11.75 (br. s, 1H). -¹³C-NMR (62.9 MHz, CDCl₃): δ=19.2, 23.6, 28.8, 33.2,36.7, 40.5, 51.6, 66.4, 67.0, 67.9, 127.7-128.6 (9×C), 134.5, 136.2,136.7, 153.5, 155.8, 163.6, 172.4. —C₃₃H₃₆N₄O₈ (616.7): calc. C, 64.27,H, 5.88; found C, 64.57, H, 6.07.

At room temperature, a solution of 300 mg (0.486 mmol) of R, methyl(E)-2-{2-[N,N′-(bisbenzyloxycarbonyl)-guanidino]ethyl-1-(benzyloxycarbonylamino)cyclo-propyl]acetatein 6 ml of dioxane is admixed with 5 ml of 2 N aqueous sodium hydroxidesolution. After 1 h, the mixture is diluted with 50 ml of water andextracted with 50 ml of ethyl acetate. By addition of 1 M hydrochloricacid and using a pH meter (glass electrode), the mixture is acidified topH=5.4 and extracted with dichloromethane. Both organic extracts are,separately, washed with in each case 30 ml of saturated ammoniumcarbonate solution and then combined and dried over magnesium sulphate.Removal of the solvent under reduced pressure gives S(E)-2-{2-[N,N′-(bisbenzyloxycarbonyl)-guanidino]ethyl-1-(benzyloxycarbonylamino)-cyclopropyl]aceticacid as an oil. -¹H-NMR (250 MHz, CDCl₃): δ=0.46 (m_(c), 1H), 1.09(m_(c), 2H), 1.61 (m_(c), 2H), 2.53 (d, J²=17 Hz, 1H), 2.75 (d, J²=17Hz, 1H), 3.4-3.8 (m, 2H), 5.05-5.20 (m, 6H), 5.83 (s, 1H), 7.2-7.5 (m,15 H), 8.60 (s, 1H), 8.97 (s, 1H), 11-12 (br. S, 1H). -FAB-MS (glycerolmatrix), m/z (%): 625 (20) [M⁺+Na], 603 (45) [M⁺+H].

The coupling of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one and acidS(E)-2-{2-[N,N′-(bisbenzyloxycarbonyl)guanidino]ethyl-1-(benzyl-oxycarbonyl-amino)-cyclopropyl]aceticacid and subsequent removal of the benzyloxycarbonyl protective groupsis carried out as described in Example 1. The title compound is obtainedas an amorphous solid (mixture of diastereomers). -¹H-NMR (250 MHz,D₂O): 0.88 (m, 1H), 0.93 (m, 1H), 1.05 (m, 1H), 1.12 (m, 2H), 2.81-3.02(m, 5H), 3.07 (m, 2H), 3.92 (m, 2H), 4.95 (m, 1H).

J 2-(2-benzyloxyethyl)-1-chloro-1-(1,2,2-trichlorovinyl)cyclopropane wasprepared according to: M. Es-Sayed, PhD-thesis, University of Hamburg1992. -M. Kordes, Diploma thesis, University of Göttingen 1996.

Example 4

(3′S,5R,S)-5-[N-Methyl-N-(3′,6′-diaminohexanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

A solution of 1.0 g (2.6 mmol) of(3S)-3-benzyloxycarbonylamino-6-tert-butyloxy-carbonylaminohexanoic acidand 5 ml of a 4 M solution of hydrogen choride in dioxane is stirred at23° C. for 30 min. The volatile components are removed under reducedpressure. This gives an oily residue which is reacted further withoutpurification.

100 mg of the residue are suspended in 3 ml of dichloromethane. 34 mg(0.316 mmol) of chlorotrimethylsilane are added. The resulting solutionis heated at 40° C. for 1 h and then cooled to 0° C. and admixedsuccessively with 45 mg (0.474 mmol) of benzyl chloroformate and 80 mg(0.789 mmol) of triethylamine. The mixture is heated at 40° C. for 1 h.0.7 ml of methanol are then added. The mixture is stirred at 23° C. for10 min, and the volatile components are then removed under reducedpressure. The residue is taken up in dichloromethane. By washing theorganic phase with 2 M hydrochloric acid, drying of the organic phasewith sodium sulphate and removal of the solvent, the residue is digestedwith diethyl ether. Filtering off and drying under reduced pressuregives 100 mg of (3S)-3,6-bis-(benzyl-oxycarbonylamino)hexanoic acid as awhite solid. ¹H-NMR (200 MHz, DMSO): 1.41 (m, 4H), 2.33 (d, 2H), 2.95(m, 2H), 3.78 (m, 1H), 5.02 (s, 2 H), 7.22 (m, 2H), 7.33 (m, 5 H). MS(DCI/NH₃): 432 (M+NH₄)⁺.

The coupling of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one and(3S)-3,6-bis(benzyloxycarbonylamino)hexanoic acid and the subsequentremoval of the benzyloxycarbonyl protective groups is carried out asdescribed in Example 1. The title compound is obtained as an amorphoussolid. ¹H-NMR (400 MHz, D₂O): 0.88 (m, 1H), 0.93 (m, 1H), 1.05 (m, 1H),1.12 (m, 2H), 2.81-3.02 (m, 5H), 3.07 (m, 2H), 3.92 (m, 2H), 4.95 (m,1H).

Example 5

(3′S,5R,S)-5-[N-Methyl-N-(3′,7′-diaminoheptanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

The coupling of 86 mg (0.47 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one and 200mg (0.47 mmol) of (3S)-3,7-bis(benzyloxycarbonyl-amino)heptanoic acidand subsequent removal of the benzyloxycarbonyl protective groups iscarried out as described in Example 1. The title compound is obtained asa white solid (57 mg, 30%). ¹H-NMR (400 MHz, CD₃OD): 1.55 (m, 2H), 1.74(m, 4H), 2.75 (m, 1H), 2.96 (m, 3H), 3.14 (m, 3H), 3.58 (m, 1H), 3.89(m, 1H), 4.01 (m, 1H), 5.14 (m, 1H).

Example 6

(3′S,5R,S)-5-{N-Methyl-N-[3′-amino-6′-(N′-methylguanidino)hexanoyl]amino}-5,6-dihydro-2-ureido-4(1H)-pyrinidoneDihydrochloride

At room temperature, a solution of 450 mg (1.1 mmol) of2′-trimethylsilylethyl (3S)-6-amino-3-benzyloxycarbonylhexanoate and 400mg (1.1 mmol) of N,N′-dibenzyl-oxycarbonyl-N-methyl S-methyl isothioureain 10 ml of DMF is admixed with 0.75 ml (5.37 mmol) of triethylamine and320 mg (1.2 mmol) of mercury(II) chloride. The mixture is stirred atroom temperature for 17 h, precipitated white solid is filtered off andthe volatile components are removed under reduced pressure. The residueis chromatographed over silica gel (dichloromethane:ethyl acetate 10:1to 3:1). This gives 470 mg (62%) of 2′-trimethylsilylethyl(3S)-3-benzyloxycarbonyl-6-[N,N′-bis-(benzyloxycarbonyl)-N-methylguanidino]hexanateas a colourless oil. MS (ESI): 705 (M+H)⁺. This product is dissolved in10 ml of THF and, at room temperature, admixed with a solution of 421 mg(1.3 mmol) of tetrabutylammonium fluoride trihydrate in 20 ml of THF.The mixture is stirred at room temperature for 2 h, and 50 ml of diethylether and 20 ml of 2 M hydrochloric acid are added. The phases areseparated and the aqueous phase is extracted with diethyl ether. Thecombined organic phases are dried over Na₂SO₄. Removal of the solventunder reduced pressure gives 250 mg (62%) of(3S)-3-benzyloxycarbonyl-6-[N,N′-bis(benzyloxycarbonyl)-N-methylguanidino]hexanoicacid as a colourless oil. MS (ESI): 605 (M+H)⁺.

The coupling of 76.5 mg (0.41 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one with theacid described and the subsequent removal of the benzyloxycarbonylprotective groups is carried out as described in Example 1. The titlecompound is obtained as a beige solid (180 mg, 99%). ¹H-NMR (400 MHz,CD₃OD): 1.70 (m, 4H), 2.72-2.94 (m, 2H), 2.84 (s, 3H), 3.14 (s, 3H),3.23 (m, 2H), 3.61 (m, 1H), 3.90 (m, 1H), 4.02 (dt, 1H), 5.15 (m, 1H).

Example 7

(3′S,5R,S)-5-[N-Methyl-N-(3′-amino-6′-ethylaminohexanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

1000 mg (3.0 mmol) of methyl(3S)-6-amino-3-benzyloxycarbonylaminohexanoate are initially charged in5 ml of 1,2-dichloroethane and, at room temperature, admixed with 250 μl(4.5 mmol) of acetaldehyde and 190 μl of acetic acid. The mixture isstirred at room temperature for 30 min and cooled to 0° C., and 1601 mg(7.6 mmol) of sodium triacetoxyborohydride are added. The mixture isstirred at room temperature for 20 h, diluted with 30 ml ofdichloromethane and extracted with 1 M hydrochloric acid. The aqueousphase is adjusted to pH 9 using sodium bicarbonate solution andextracted three times with in each case 30 ml of ethyl acetate. Thecombined organic phases are dried using Na₂SO₄ and the solvent isdistilled off under reduced pressure. This gives 640 mg (66%) of methyl(3S)-3-benzyloxy-carbonylamino-6-ethylaminohexanoate as a colourlessoil. ¹H-NMR (200 MHz, DMSO): 0.97 (t, 3H), 1.37 (m, 4H), 2.42 (m, 6H),3.56 (s, 3H), 3.78 (m, 1H), 5.02 (s, 2H), 7.35 (m, 6H). MS (DCI/NH₃):323 (M+H)⁺.

The product described (630 mg, 1.95 mmol) is dissolved in 10 ml ofdichloromethane and, at 0° C., admixed with 300 μl (2.15 mmol) oftriethylamine and 310 μl (2.15 mmol) of benzyl chloroformate. Themixture is stirred at room temperature for 16 h, the organic phase iswashed twice with water and dried over Na₂SO₄ and the solvent is removedunder reduced pressure. The residue is chromatographed over silica gel(ethyl acetate/cyclohexane 1:1). This gives 515 mg (58%) of methyl(3S)-3-benzyloxycarbonylamino-6-[(benzyloxycarbonyl)ethyl-amino]hexanoateas a white solid. ¹H-NMR (200 MHz, DMSO): 1.02 (t, 3H), 1.40 (m, 4H),2.42 (m, 2H), 3.18 (m, 4H), 3.56 (s, 3H), 3.82 (m, 1H), 5.01 (s, 2H),5.06 (s, 2H), 7.25 (m, 1H), 7.35 (m, 10H). MS (ESI): 457 (M+H)⁺.

The product described (510 mg, 1.12 mmol) is dissolved in 4 ml ofdichloromethane and, at room temperature, mixed with 158 mg (1.30 mmol)of potassium trimethylsilanolate. The mixture is stirred at roomtemperature for 16 h, diluted with 20 ml of dichloromethane and washedwith 1 M of hydrochloric acid, the organic phase is dried over Na₂SO₄and the volatile components are removed under reduced pressure. Thisgives 463 mg (94%) of(3S)-3-benzyloxycarbonylamino-6-[(benzyloxycarbonyl)-ethyl]aminohexanoicacid as a white solid. ¹H-NMR (200 MHz, DMSO): 1.02 (t, 3H), 1.40 (m,4H), 2.35 (m, 2H), 3.20 (m, 4H), 3.81 (m, 1H), 5.00 (s, 2H), 5.05 (s,2H), 7.25 (m, 1H), 7.33 (m, 10H). MS (ESI): 443 (M+H)⁺.

The coupling of 42 mg (0.27 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one and 100mg (0.27 mmol) of the acid described and the subsequent removal of thebenzyloxycarbonyl protective groups is carried out as described inExample 1. The title compound is obtained as a white solid (60 mg, 64%).¹H-NMR (400 MHz, CD₃OD): 1.32 (t, 3H), 1.83 (m, 4H), 2.80 (dd, 1H),2.95-3.18 (m, 5H), 3.19 (s, 3H), 3.61 (m, 1H), 3.90 (ddd, 1H), 4.03 (dt,1H), 5.18 (m, 1H). MS (ESI): 342 (M+H)⁺.

Example 8

(3′S,5R,S)-5-[N-Methyl-N-(3′,5′-diaminopentanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

The coupling of 46 mg (0.25 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one and 100mg (0.25 mmol) of (3S)-3,5-bis-(benzyloxy-carbonylamino)pentanoic acidand the subsequent removal of the benzyloxycarbonyl protective groups iscarried out as described in Example 1. The title compound is obtained asa white solid. (25 mg, 27%). ¹H-NMR (400 MHz, CD₃OD): 2.10 (m, 2H), 2.86(dd, 1H), 3.04 (m, 1H), 3.10 (dd, 2H), 3.18 (s, 3H), 3.72 (m, 1H), 3.89(ddd, 1H), 4.02 (dt, 1H), 5.19 (m, 1H).

Example 9

(3′R,5R,S)-5-[N-(3′-amino-6′-guanidinohexanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

The preparation of the title compound and the required component(5R,S)-3,4,5,6-tetrahydro-5-amino-2-ureidopyrimidin-4-one were carriedout analogously to published syntheses (cf. V. V. Sokolov, S. I.Kozhushkov, S. Nikolskaya, V. N. Belov, M. Es-Sayed, A. de Meijere, Eur.J. Org. Chem. 1998, 777). ¹H-NMR (200 MHz, D₂O): 1.45-1.65 (m, 4H),2.55-2.70 (m, 2H), 3.05-3.13 (m, 2H), 3.55 (m, 1H), 3.62 (dd, 1H), 3.71(dd, 1H), 4.87 (dd, 1H).

Example 10

(3′S,5R,S)-5-[N-Ethyl-N-(3′,6′-diaminohexanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

The coupling of 120 mg (0.60 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-ethylamino-2-ureidopyrimidin-4-one(synthesized analogously to the methyl compound: cf. V. V. Sokolov, S.I. Kozhushkov, S. Nikolskaya, V. N. Belov, M. Es-Sayed, A. de Meijere,Eur. J. Org. Chem. 1998, 777) and 250 mg (0.60 mmol) of(3S)-3,6-bis-(benzyloxy-carbonylamino)hexanoic acid and the subsequentremoval of the benzyloxycarbonyl protective groups are carried out asdescribed in Example 1. The title compound is obtained as an amorphoussolid (115 mg, 48%). ¹H-NMR (400 MHz, CD₃OD): 1.26 (t, 3H), 1.78 (m,4H), 2.62-2.90 (m, 2H), 2.98 (m, 2H), 3.49 (m, 1H), 3.63 (m, 2H), 3.89(m, 1H), 4.08 (m, 1H), 4.62 (m, 1H). MS (ESI): 328 (M+H)⁺.

Example 11

(4′S,5R,S)-5-[N-Methyl-N-(4′,7′-diaminoheptanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

(4S)4,7-Bis-(benzyloxycarbonylamino)heptanoic acid was synthesized from(3S)-3,6-bis-(benzyloxycarbonylamino)hexanoic acid (cf. Example 4)analogously to a literature example (H. M. M. Bastiaans, A. E.Alewijnse, J. L. van der Baan, H. C. J. Ottenheijm, Tetrahedron Lett.1994, 35, 7659). The coupling of 22 mg (0.12 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one and 50mg (0.12 mmol) of the corresponding acid and the subsequent removal ofthe benzyloxycarbonyl protective groups are carried out as described inExample 1. The title compound is obtained as a white solid (25 mg, 52%).MS (ESI): 328 (M+H)⁺.

Example 12

(3′R,5R,S)-5-[N-Methyl-N-(3′,6′-diaminohexanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

The coupling of 112 mg (0.60 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one and 250mg (0.60 mmol) of (3R)-3,6-bis-(benzyloxycarbonyl-amino)hexanoic acidand the subsequent removal of the benzyloxycarbonyl protective groupsare carried out as described in Example 1. The title compound isobtained as a beige solid (204 mg, 88%). ¹H-NMR (400 MHz, CD₃OD): 1.78(m, 4H), 2.80 (m, 2H), 2.96 (m, 2H), 3.17 (s, 3H), 3.62 (m, 1H), 3.92(m, 1H), 4.03 (n, 1H), 5.18 (m, 1H).

Example 13

(3′R,5R,S)-5-[N-Methyl-N-(3′-amino-5′-carbamoyl-pentanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

The coupling of 157 mg (0.85 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one and 250mg (0.85 mmol) of (3R)-3-(benzyloxycarbonyl-amino)-5-carbamoylpentanoicacid and the subsequent removal of the benzyloxycarbonyl protectivegroups are carried out as described in Example 1. The title compound isobtained as a beige solid (81 mg, 26%). ¹H-NMR (400 MHz, CD₃OD): 1.94(m, 2H), 2.45 (m, 2H), 2.72 (m, 1H), 2.91 (m, 1H), 3.09 (s, 3H), 3.61(m, 1H), 3.78 (ddd, 1H), 3.94 (m, 1H), 5.15 (m, 1H).

Example 14

(3′R,5R,S)-5-[N-(3′,6′-Diaminohexanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

The coupling of 62 mg (0.36 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-amino-2-ureido-pyrimidin-4-one (cf. Example10) and 150 mg (0.36 mmol) of(3S)-3,6-bis-(benzyl-oxycarbonylamino)hexanoic acid and the subsequentremoval of the benzyloxy-carbonyl protective groups are carried out asdescribed in Example 1. The title compound is obtained as a white solid(110 mg, 82%). ¹H-NMR (400 MHz, CD₃OD): 1.82 (m, 4H), 2.75 (dd, 1H),2.80 (dd, 1H), 2.99 (m, 2H), 3.62 (m, 1H), 3.78 (m, 1H), 3.94 (m, 1H),5.02 (m, 1H).

Example 15

(3′S,5R,S)-5-[N-Ethyl-N-(3′-amino-6′-guanidinohexanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

The synthesis of(5R,S)-3,4,5,6-tetrahydro-5-ethylamino-2-ureidopyrimidin-4-one and thereaction of this building block with(3S)-1-diazo-3-benzyloxycarbonyl-6-[N,N′-bis-(benzyloxycarbonyl)guanidino]hexan-2-oneare carried out analogously to a published procedure (cf. V. V. Sokolov,S. I. Kozhushkov, S. Nikolskaya, V. N. Belov, M. Es-Sayed, A. deMeijere, Eur. J. Org. Chem. 1998, 777). The starting material used isN-ethyl-DL-asparagine (Y. Liwschitz, Y. Edlitz-Pfeffermann, Y. Lapidoth,J. Am. Chem. Soc. 1956, 78, 3069). The subsequent removal of thebenzyloxycarbonyl protective groups is carried out as described forExample 1. The title compound is obtained as a white solid: meltingpoint: 170-172° C. ¹H-NMR (250 MHz, D₂O): 1.03 (t, 3H), 1.35-1.55 (m,4H), 2.25-2.45 (m, 2H), 2.95-3.05 (m, 2H), 3.30-3.77 (m, 5H), 4.42 (m,1H). MS (FAB): 370 (M+H)⁺.

Example 16

(3′S,5R,S)-5-[N-Methyl-N-(3′-amino-6′-methoxycarbonylaminohexanoyl)-amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneHydrochloride

The coupling of 104 mg (0.56 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one and 190mg (0.56 mmol) of(3S)-3-benzyloxycarbonylamino-6-methoxycarbonylaminohexanoic acid andthe subsequent removal of the benzyloxycarbonyl protective group arecarried out as described in Example 1. The title compound is obtained asa white solid (30 mg, 13%). ¹H-NMR (400 MHz, CD₃OD): 1.58 (m, 2H), 1.69(m, 2H), 2.65-3.07 (m, 4H), 3.14 (m, 3H), 3.57 (m, 1H), 3.63 (s, 3H),3.88 (m, 1H), 4.03 (m, 1H), 5.18 (m, 1H). MS (ESI): 372 (M+H)⁺.

Example 17

(3′R,S,5R,S)-5-[N-Methyl-N-(3′,8′-diaminooctanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneDihydrochloride

3.70 g (14.7 mmol) of 6-benzyloxycarbonylamino-1-hexanol (S. Fernandez,E. Menendez, V. Gotar, Synthesis 1991, 713-716) and 14.9 g (147 mmol) oftriethylamine are dissolved in 50 ml of dichloromethane. The solution iscooled to 0° C. and admixed with 7.03 g (44.2 mmol) of sulphurtrioxide/pyridine complex in 44 ml of dimethyl sulphoxide. The mixtureis then warmed to room temperature and stirred for 25 min. The solutionis poured into 400 ml of ice-water and extracted repeatedly with diethylether. The combined organic phases are washed three times with 1 Mhydrochloric acid and once each with water and saturated aqueous NaClsolution, dried over Na₂SO₄ and freed from the solvent under reducedpressure. The resulting colourless oil (3.50 g) is dissolved in 20 ml ofTHF (solution A). Separately, 3.54 g (16.9 mmol) of methyldiethylphosphonoacetate in 40 ml of THF are admixed, at 0° C., with 17ml of a 1 M THF solution of sodium (bistrimethylsilyl)amide. The mixtureis stirred for 45 min, and solution A is added at 0° C. The resultingsolution is warmed to room temperature, stirred for 2 h and concentratedunder reduced pressure. The residue is chromatographed over silica gel(ethyl acetate/cyclohexane 1:4 to 1:2). This gives 1.73 g (34%) ofmethyl (Z)-8-benzyloxycarbonylamino-2-octenecarboxylate as a colourlessoil. ¹H-NMR (200 MHz, DMSO): 1.17-1.49 (m, 6H), 2.18 (q, 2H), 2.95 (q,2H), 3.66 (s, 3H), 5.00 (s, 2H), 5.87 (d, 1H), 6.89 (td, 1H), 7.25 (m,1H), 7.34 (m, 5H). MS (DCI/NH₃): 323 (M+NH₄)⁺.

0.88 g (2.9 mmol) of the ester is added to 9 ml of a solution of ethanolsaturated with ammonia. In a closed vessel, the mixture is heated at100° C. (bath temperature) for 6 h. After cooling to room temperature,the volatile components are removed under reduced pressure. The residueis taken up in 15 ml of dichloromethane. The resulting solution iscooled to 0° C. and admixed successively with 0.58 ml (4.2 mmol) oftriethylamine and 0.51 ml (3.6 mmol) of benzyl chloroformate. Themixture is allowed to warm to room temperature and stirred for another15 h. The mixture is diluted with 50 ml of dichloromethane and washedwith 1 M of hydrochloric acid, the organic phase is dried over Na₂SO₄and the volatile components are removed under reduced pressure.Chromatography of the residue over silica gel (ethyl acetate/cyclohexane1:3 to 1:2) gives 194 mg (14%) of ethyl(3R,S)-3,8-bis(benzyloxycarbonylamino)octanoate [MS (ESI): 471 (M+H)⁺]and 248 mg (19%) of methyl(3R,S)-3,8-bis(benzyloxycarbonylamino)octanoate [MS (ESI): 457 (M+H)⁺]in the form of colourless oils. Both products are combined, dissolved in10 ml of dichloromethane and admixed with 280 mg (1.9 mmol) of potassiumtrimethylsilanolate. The mixture is stirred at RT for 1 h, a further 100mg of potassium trimethylsilanolate are added and stirring is continuedfor another hour. The mixture is diluted with 20 ml of dichloromethane,the organic phase is washed with 2 M hydrochloric acid and dried overNa₂SO₄ and the solvent is evaporated under reduced pressure. This gives393 mg (93%) of (3R,S)-3,8-bis(benzyloxy-carbonylamino)octanoic acid asa white solid. ¹H-NMR (300 MHz, DMSO): 1.21 (m, 4H), 1.38 (m, 4H), 2.36(m, 2H), 2.95 (q, 2H), 3.77 (m, 1H), 5.01 (s, 4H), 7.14 (m, 1H), 7.35(m, 10H), 12.08 (s, 1H).

The coupling of 200 mg (0.45 mmol) of the acid thus prepared with 84 mg(0.45 mmol) of(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one and thesubsequent removal of the benzyloxycarbonyl protective groups arecarried out as described in Example 1. The title compound is obtained asa white solid (175 mg, 94%). ¹H-NMR (400 MHz, CD₃OD): 1.47 (m, 4H), 1.72(m, 4H), 2.75 (m, 1H), 2.94 (m, 3H), 3.15 (m, 3H), 3.58 (m, 1H), 3.90(m, 1H), 4.03 (m, 1H), 5.18 (m, 1H). MS (ESI): 342 (M+H)⁺.

Example 18

(3′R,S,5R,S)-5-[N-Methyl-N-(3′-amino-5′-cyanopentanoyl)amino]-5,6-dihydro-2-ureido-4(1H)-pyrimidoneHydrochloride

A solution of 1.00 g (10.1 mmol) of the sodium salt of 3-cyanopropanoicacid in 50 ml of dichloromethane was extracted with 30 ml of a 1-molarhydrochloric acid. The organic phase was dried over magnesium sulphateand the solvent was distilled off using a rotary evaporator. The crude3cyanopropanoic acid was freed of solvent residues under high vacuum.

The residue was taken up in 15 ml of THF and, at 0° C., admixed a littleat a time with 1.96 g (12.1 mmol) of N,N-carbonyldiimidazole. Themixture was stirred at room temperature for one hour (solution A).

In a second flask, a solution of 960 mg (10.1 mmol) of magnesiumchloride and 2.75 g (15.1 mmol) of the potassium salt of ethyl malonatein 25 ml of THF was heated at 50° C. for 4 hours. The mixture was cooledto room temperature and then admixed dropwise with the solution A whichhad been prepared earlier. The mixture was stirred at room temperatureovernight.

The solvent was distilled off using a rotary evaporator and the residuewas taken up in 20 ml of water and 50 ml of dichloromethane. The organicphase was filtered through kieselguhr and dried over sodium sulphate.The residue was purified over a flash column (silica gel, mobile phasecyclohexane/ethyl acetate 10:1 with increasing polarity to 1:1). Thisgave 617 mg (36%) of ethyl 5-cyano-3-oxopentanoate. ¹H-NMR (300 MHz,DMSO) 1.19 (t, 3H), 2.60 (t, 2H), 2.95 (t, 2H), 3.62 (s, 2H), 4.10 (q,2H). MS (EI): 169 (M)⁺.

3.80 g (22.4 mmol) of ethyl 5-cyano-3-oxopentanoate were taken up in 5ml of a saturated ethanolic ammonia solution and stirred at roomtemperature for 24 h. The volatile components were distilled off using arotary evaporator, giving 3.60 g (95%) of ethyl3-amino-5-cyano-2-pentenoate. ¹H-NMR (300 MHz, DMSO) 1.16 (t, 3H), 2.37(t, 2H), 2.75 (t, 2H), 3.99 (q, 2H), 4.41 (s, 1H), 6.96 (s, broad, 1H),7.69 (s, broad, 1H). MS (DCI/NH₃): 169 (M+H)⁺, 186 (M+NH₄)⁺, 337(2M+H)⁺.

At 0° C., a solution of 50.0 mg (297 μmol) of ethyl3-amino-5-cyano-2-pentenoate in 1 ml of methanol was added dropwise to asolution of 56.0 mg (892 μmol) of sodium cyanoborohydride in 1 ml ofabs. methanol. The mixture was mixed with 6 drops of glacial aceticacid, the cooling bath was removed and the mixture was stirred at roomtemperature for 2 h.

The mixture was admixed with 1 ml of a sat. sodium bicarbonate solutionand concentrated using a rotary evaporator. The aqueous phase wasextracted twice with in each case 5 ml of dichloromethane. The organicphase was dried over sodium sulphate and the solvent was distilled offusing a rotary evaporator. 39.9 mg (79%) of the desired ethyl3-amino-5-cyanopentanoate remained. ¹H-NMR (300 MHz, DMSO) 1.19 (t, 3H),1.49 (m, 1H), 1.68 (m, 1H), 2.27 (dd, 1H), 2.40 (dd, 1H), 2.55 (t, 2H),3.00 (m, 1H), 4.08 (q, 2H). MS (DCI/NH₃): 171 (M+H)⁺.

At room temperature, a solution of 723 mg (3.31 mmol) of BOC anhydridein 0.5 ml of dioxane was added dropwise to a solution of 470 mg (2.76mmol) of ethyl 3-amino-5-cyanopentanoate and 458 mg (3.31 mmol) ofpotassium carbonate in 10ml of dioxane/water (1:1). The volatilecomponents were distilled off using a rotary evaporator and the residuewas extracted twice with in each case 5 ml of dichloromethane. Theorganic phase was dried over sodium sulphate and the solvent wasdistilled off using a rotary evaporator. The crude product was purifiedover a flash column (silica gel, mobile phase:cyclohexane/ethyl acetate20:1 with increasing polarity to 1:1). This gave 505 mg (68%) of ethyl3-[(tert-butoxycarbonyl)amino]-5-cyanopentanoate. ¹H-NMR (300 MHz, DMSO)1.19 (t, 3H), 1.35 (s, 9H), 1.70 (m, 2H), 2.48 (m, 4H), 3.81 (m, 1H),4.02 (q, 2H), 6.80 (m, 1H). MS (DCI/NH₃): 288 (M+NH)⁺.

213 mg (1.66 mmol) of potassium trimethylsilanolate were added to asolution of 300 mg (1.11 mmol) of ethyl3-[(tert-butoxycarbonyl)amino]-5-cyanopentanoate in 1 ml ofdichloromethane, and the mixture was stirred at room temperature. After2 hours, another 213 mg of potassium trimethylsilanolate were added, andthe mixture was stirred for 30 min.

The mixture was admixed with 1 ml of a sat. ammonium chloride solutionand extracted with 2 ml of dichloromethane. The aqueous phase wasadjusted to pH 1 using 1-molar hydrochloric acid and extracted twicewith in each case 3 ml of dichloromethane. The combined organic phaseswere dried over sodium sulphate and freed from the solvent using arotary evaporator. This gave 177 mg (66%) of the desired3-[(tert-butoxycarbonyl)amino]-5-cyanopentanoic acid. ¹H-NMR (300 MHz,d⁶-DMSO) 1.38 (s, 9H), 1.65 (m , 1H), 1.75 (m, 1H), 2.38 (m, 2H), 2.45(m, 2H), 3.79 (m, 1H), 6.80 (d, 1H), 12.20 (s, broad, 1H). MS (DCI/NH₃):260 (M+NH₄)⁺.

The coupling of 15 mg (82 μmol) of3-[(tert-butoxycarbonyl)amino]-5-cyano-pentanoic acid with(5R,S)-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-one wascarried out as described in Example 1. The yield was 32%. To remove theBOC group, the crude product was taken up in 1 ml of 4 molar HCl indioxane and stirred at room temperature for 30 min. All volatilecomponents were distilled off using a rotary evaporator. The residue wastaken up in methanol and admixed dropwise with acetone until aprecipitate was formed. The supernatant was decanted off and the whitesolid that remained was freed from the solvent residues under oilpumpvacuum. This gave 2.1 mg (28%) of the title compound. MS (DCI): 311(M+H)⁺.

What is claimed is:
 1. Compounds of the formula:

in which R¹ represents hydrogen or (C₁-C₆)alkyl, X represents a group ofthe formula —(CH₂)_(m)—, in which m is 1 or 2, D is selected from groupsof the formulae D₁ to D₃

 in which R² represents hydrogen or hydroxyl, R³ represents hydrogen, orR² and R³ together form an oxo group, Y represents a straight-chain orbranched (C₁-C₅)alkanediyl group which may optionally be substituted byhydroxyl or oxo, or represents a group of the formula below

 in which r and s are identical or different and are 0, 1 or 2, Zrepresents a group selected from groups of the formulae

in which R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷,R¹⁸ and R¹⁹ in each case independently of one another are selected fromthe group consisting of hydrogen, (C₁-C₆)alkyl, (C₁-C₄)alkanoyl,t-butoxycarbonyl, benzyloxycarbonyl and benzyl, Q represents oxygen orsulphur and p is 1, 2 or 3 and with the proviso, that R¹ does notrepresent methyl, m does not represent 1, D does not represent D₁ and Ydoes not represent —(CH₂)₃— when Z represents a group of the formula

and pharmaceutically acceptable salts thereof.
 2. Compounds according toclaim 1 of the formula (I), in which R¹ represents hydrogen or(C₁-C₆)alkyl, X represents a group of the formula —(CH₂)_(m)—, in whichm is 1 or 2, D is selected from groups of the formulae D₁ to D₃

 in which R² represents hydrogen or hydroxyl, R³ represents hydrogen, orR² and R³ together form an oxo group, Y represents a straight-chain orbranched (C₁-C₅)alkanediyl group which may optionally be substituted byhydroxyl or oxo, or represents a group of the formula below

 or different and are 0, 1 or 2, Z represents a group selected fromgroups of the formulae

 in which R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶,R¹⁷, R¹⁸ and R¹⁹ in each case independently of one another are selectedfrom the group consisting of hydrogen, (C₁-C₆)alkyl, (C₁-C₄)alkanoyl,carbonyl and benzyl, Q represents oxygen or sulphur and pis 1, 2 or 3and with the proviso, that R¹ does not represent methyl, m does notrepresent 1, D does not represent D₁ and Y does not represent —(CH₂)₃—when Z represents a group of the formula

and pharmaceutically acceptable salts thereof.
 3. Compounds according toclaim 1 in which m is 1 or
 2. 4. Compounds according to claim 1, inwhich Y represents a straight-chain or branched (C₁-C₅)alkanediyl group.5. Compounds according to claim 1, in which Y represents a group of theformula

in which r and s are as defined above in claim
 1. 6. Compounds accordingto claim 1, in which Y represents m-phenylene.
 7. Compounds according toclaim 1, in which r and s are
 0. 8. Compounds according to claim 1, inwhich Z represents a group of the formula

in which R¹⁸ and R¹⁹ are as defined in claim
 1. 9. Compounds accordingto claim 1, in which Z represents a group of the formulae

in which R⁴, R⁵, R⁶, R⁷ and R¹⁷ are as defined in claim
 1. 10. Compoundsaccording to claim 1, in which Z represents a group of the formula

in which R⁴, R⁵, R⁶ and R⁷ are as defined in claim
 1. 11. Compoundsaccording to claim 1, in which D represents a group of the formula


12. Compounds according to claim 1, in which D represents a group of theformula


13. Compounds according to claim 1, in which D represents a group of theformula


14. Compound according to claim 1 of the formula

and pharmaceutically acceptable salts thereof.
 15. Compound according toclaim 1 of the formula

and pharmaceutically acceptable salts thereof.
 16. Process for preparingcompounds according to claim 1, characterized in that compounds of theformula (II)

in which X, Y and Z are as defined in claim 1 and D′ is selected fromgroups of the formulae D′1 to D′3

in which R² and R³ are as defined in claim 1 and A is a protected aminogroup are reacted with compounds of the formula (III)

in which R¹ is as defined in claim 1, in the presence of couplingagents, and the protective group on the protected amino group A isremoved.
 17. Process according to claim 16, in which the coupling agentis selected from O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) or bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBroP).
 18. Pharmaceutical composition, comprisinga compound according to claim 1 and a pharmaceutically acceptablecarrier or excipient.
 19. A method of treating an infection bygram-positive bacteria, gram-negative bacteria and corynebacteria inhumans or animals, comprising administering to a human or animal aneffective amount of a compound according to claim 1.